Mitochondria play a vital role in cell physiology and the response to environmental stress. A number of cellular toxins, including rotenone, 1-methyl-4-phenylpyridine (MPP+) and paraquat act to impair mitochondrial electron transport, generating ROS. Reactive oxygen species are also important in the toxicity of arsenic, amyloid, and ceramide. Mitochondria have been viewed as a potential source of ROS that may contribute to Parkinson's disease, aging and other pathological conditions. Since mitochondria contain only a small 16.5 kb mtDNA genome, encoding only 13 proteins, the organelle depends on the nucleus for most gene products, including all of the factors required for DNA replication, expression and repair. Recent studies from our laboratory and others have revealed an increasing collection of proteins that function in both mitochondria and other cellular compartments. A number of these proteins function in repair of oxidative damage to mtDNA. One significant consequence of mitochondrial pathology is the generation of mutations in mtDNA, many of which have a tissue-specific incidence, occurring most commonly in postreplicative tissues such as nerve and muscle. The investigators propose to test the hypothesis that mitochondria in differentiated cells may contain a different complement of proteins than actively dividing cells which may predispose post-replicative cells to a higher rate of mtDNA mutations or may alter the ability of cells to enter apoptosis. To accomplish this, they will study the effect of oxidative stress on the mitochondrial proteome in both embryonal carcinoma cells that are actively dividing and in cells that have been induced to differentiate along a neuronal pathway. Both 2-D gel methods and quantitative isotope-coded affinity tag (ICAT) methods will be used to compare the abundance of mitochondrial proteins in control cells and cells exposed to oxidative stress. The broad proteomic screen will permit the discovery of novel gene products not previously known to function in mitochondria. Data will be analyzed to provide new insights into networks of proteins acting to repair oxidative damage to mtDNA or to detoxify ROS.